Bacteria use quorum sensing (QS) to induce specific phenotypes as a function of population density. In Gram-negative bacteria, this QS process is largely driven by the detection of structurally distinct N-acylated L- homoserine lactones (AHLs). In the simplest Gram-negative QS systems, bacteria produce AHLs that diffuse across the bacterial membrane. At high cell densities, intracellular AHL concentrations are sufficient to bind its cognate receptor, induce the formation of transcriptionally active receptor dimers, and thereby promote the transcription of target genes. Many of these target genes promote bacterial virulence, therefore, the small molecule disruption of bacterial QS is an attractive antivirulence strategy. The QS system of P. aeruginosa is a particularly attractive target for this strategy, as this pathogen is prevalent in the lungs of cystic fibrosis patients, is resistant to most traditionl antibiotics, and employs QS to regulate virulence factor production and biofilm formation. However, the P. aeruginosa QS system is also very complex, as it employs three receptors, the activities of which are intimately interconnected, and two AHLs, one of which is recognized by two receptors. The complexity of this QS circuit has prevented its complete characterization and thereby hampered its exploitation as a target for antivirulence agents. Therefore, efforts aimed at obtaining a greater understanding of the receptor interactions that govern QS regulation of P. aeruginosa virulence are warranted. This research training plan describes our intent to demonstrate the application of non-native AHLs as chemical tools for the characterization of the P. aeruginosa QS circuit. Our first aim is to identify non-native AHLs that modulate P. aeruginosa QS in a receptor-selective manner. This will be accomplished by using QS reporter strains of P. aeruginosa and E. coli with similar receptor expression levels to screen a library of synthetic AHLs for receptor regulation. AHLs displaying receptor-selective activities will then be applied as chemical tools to characterize the P. aeruginosa QS circuit. The first of these characterization efforts will involve the use of AHL chemical tools to modulate the activities of individual receptors in the P. aeruginosa QS circuit. Both virulence factor assays and transcriptional profiling can be used to monitor the effects of individual perturbations of the QS circuit. This will characterize the extent to which each receptor contributes to P. aeruginosa virulence and will facilitate the delineation of receptor hierarchy in this pathogen. The second characterization effort involves the application of AHL chemical tools to study the temporal nature by which receptors are activated in the P. aeruginosa QS circuit. The temporal response of the P. aeruginosa QS system to activation of each receptor will be monitored using reporter strains of receptor activity. This characterization can be applied to the development of new AHL dosing strategies for the treatment of P. aeruginosa infection. These efforts will demonstrate the utility of chemical tools for studying QS circuits and will facilitate the development of antivirulence strategies for the treatment of P. aeruginosa infections. PUBLIC HEALTH RELEVANCE: Bacteria use chemical language to induce behaviors that promote the establishment, proliferation, and persistence of infection. This research aims to characterize the process by which this language is translated and perceived by P. aeruginosa. The information gained from this study will facilitate the development of treatments for P. aeruginosa infections.